pepstatin a  (Millipore)

Journal: Oncotarget

Article Title: Regulation of the antiapoptotic protein cFLIP by the glucocorticoid Dexamethasone in ALL cells

doi: 10.18632/oncotarget.24782

Figure Lengend Snippet: Dexa-stimulated downregulation of cFLIP L protein occurs largely independent of the proteasome, lysosomal enzymes and caspases ( A ) Molt-4 and Reh cells were treated with 30 nM Bortezomib to block proteasomal activity, after one hour cells were treated with 10 ng/ml CHX for indicated time points. Expression of cFLIP L and Noxa was analyzed by Western blotting. β-Actin served as loading control. ( B ) Molt-4 and Reh cells were treated with 30 nM Bortezomib (Bort) to block proteasomal activity, after one hour cells were treated with 300 µM Dexa for four hours. Expression of cFLIP L was analyzed by Western blotting. GAPDH served as loading control. ( C ) Proteasome activity was analyzed using Chemicon 20S Proteasome Activity Assay. Fold change of proteasome activity of two independent experiments performed in duplicates is shown. EtOH was used as solvent for Dexa. ( D ) Reh cells were incubated with inhibitors of lysosomal enzymes (10 µg/ml E64d, 10 µg/ml CA-074 methyl ester (CA), 10 µg/ml Pepstatin A (Pep A), 50 nM Bafilomycin A (Baf), 25 µM Chloroquine (CQ)) for one hour, followed by treatment with 300 µM Dexa for four hours. cFLIP L protein expression was analyzed using Western blotting. β-Actin served as loading control. ( E ) Jurkat and Reh cells were treated with 20 µM zVAD.fmk and 300 µM Dexa for four hours or indicated time points. cFLIP L expression was analyzed by Western blotting. GAPDH served as loading control.

Article Snippet: The glucocorticoid Dexa was purchased from Sigma-Aldrich (Steinheim, Germany), the caspase inhibitor zVAD.fmk from Bachem (Heidelberg, Germany), the proteasome inhibitor Bortezomib from Selleckchem (Houston, TX, USA) and cycloheximide (CHX), E64d, CA-074 methyl ester, chloroquine, pepstatin A and Bafilomycin A from Sigma-Aldrich.

Techniques: Blocking Assay, Activity Assay, Expressing, Western Blot, Incubation

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism

doi: 10.1128/JVI.00191-18

Figure Lengend Snippet: pUL38 inhibits cathepsin leakage-mediated cell death. (A) MRC5 cells were infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell samples were collected 72 h later. Cytosolic proteins were separated and analyzed by immunoblotting with the indicated antibodies. (B) MRC5 cells were treated with Tiron (200 μM), CPX (0.5 μM), or DMSO and then infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell samples were collected 72 h later. Cytosolic proteins were separated and analyzed by immunoblotting with the indicated antibodies. (C) MRC5 cells were treated with pepstatin A (pep A) (25 μM) plus E64d (10 μM) or with DMSO and then infected with wild-type or pUL38-deficient HCMV at an MOI of 3. Cell viability was assessed at the indicated time points by CellTiter-Glo luminescent cell viability assay. The viability of cells infected with wild-type HCMV infected cells was set as 100%, and the viability of cells infected with pUL38-deficient HCMV was normalized to that of wild-type-HCMV-infected cells. Data shown represent the mean ± SD from three independent experiments. ***, P

Article Snippet: The iron chelators Tiron and ciclopirox olamine (CPX), the ROS scavengers Trolox and N -acetyl- l -cysteine (NAC), the ER stress inducers tunicamycin (TM) and thapsigargin (TG), the cathepsin inhibitors pepstatin A and E64d, and the eukaryote translation inhibitor cycloheximide (CHX) were purchased from Sigma-Aldrich.

Techniques: Infection, Cell Viability Assay

Journal: Journal of Cancer

Article Title: Lycopene upregulates ZO-1 and downregulates claudin-1 through autophagy inhibition in the human cutaneous squamous cell carcinoma cell line COLO-16

doi: 10.7150/jca.26578

Figure Lengend Snippet: Cells were treated with 0, 5, 10 or 20 μM lycopene (a) with or without 10 μg/mL E64d and 10 μg/mL pepstatin (b) for 24 hours. Western blotting analysis was performed using a primary antibody against LC3A/B. GAPDH served as a loading control. The LC3-II / LC3-I and LC3-II / GAPDH ratios were calculated. Cells were treated with or without 10 μM lycopene in the presence or absence of 10 μg/mL E64d and 10 μg/mL pepstatin for 24 hours (c and d). Cells stained by AO (c) or transfected with GFP-LC3B (d) were visualized using a laser scanning confocal microscope. The means of the red/green fluorescence ratios (c) or the GFP-LC3 puncta (d) for individual cells were determined for statistical analysis. The data are shown as the means ± SD of three independent experiments, and representative figures are shown. Bars = 5 μm. *: p

Article Snippet: Reagents and antibodies The compounds used in this study included lycopene (sc-205738, Santa Cruz Biotechnology, Dallas, TX, USA) dissolved in tetrahydrofuran (0.1% was used in the non-treatment cells as solvent control), rapamycin, trehalose, E64d, pepstatin, acridine orange (AO), and dimethyl sulfoxide (DMSO) (all from Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot, Staining, Transfection, Microscopy, Fluorescence

Journal: Journal of Cancer

Article Title: Lycopene upregulates ZO-1 and downregulates claudin-1 through autophagy inhibition in the human cutaneous squamous cell carcinoma cell line COLO-16

doi: 10.7150/jca.26578

Figure Lengend Snippet: Cells were treated with 0, 5, 10 or 20 μM lycopene (a), and then the protein levels and phosphorylation of MTOR, p70 S6 kinase, ribosomal protein S6 and 4E-BP1 were determined by western blotting. Cells were treated with or without 10 μM lycopene in the presence or absence of 80 nM rapamycin (b and c), 1 μM torin 1 (d) or 100 mM trehalose (f) for 24 hours, and the protein levels and phosphorylation of MTOR (b, d and f) and ribosomal protein S6 (b), as well as the protein levels of ZO-1, claudin-1 (c, d and f) and LC3 (d and f), were determined by western blotting. In addition, COLO-16 cells were treated with or without 10 μM lycopene in the presence or absence of 10 μg/mL E64d and 10 μg/mL pepstatin or 80 nM rapamycin (e), and the levels of LC3 protein were determined by western blotting. GAPDH served as a loading control. The LC3-II / LC3-I or LC3-II / GAPDH ratios were calculated. The data are shown as the means ± SD of three independent experiments, and representative figures are shown. *: p

Article Snippet: Reagents and antibodies The compounds used in this study included lycopene (sc-205738, Santa Cruz Biotechnology, Dallas, TX, USA) dissolved in tetrahydrofuran (0.1% was used in the non-treatment cells as solvent control), rapamycin, trehalose, E64d, pepstatin, acridine orange (AO), and dimethyl sulfoxide (DMSO) (all from Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot

Journal: PLoS Pathogens

Article Title: Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

doi: 10.1371/journal.ppat.1003809

Figure Lengend Snippet: Blockade of Akt induces accumulation of the autophagy protein LC3 around the parasite, vacuole-lysosome fusion and killing of T. gondii dependent on the autophagy proteins. A , HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy 2 h and 24 h post-challenge. B , HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge. C , RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy 2 h and 24 h post-challenge. D , mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IV followed by challenge with T. gondii -RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite. E , HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole. F , Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii -YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite G, H , mHEVc cells were transfected with Beclin 1 siRNA ( G ), Atg7 siRNA ( H ) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii . Monolayers were examined by light microscopy at 24 h. I , mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii . 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge. J , Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii . Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.

Article Snippet: In certain experiments, mammalian cells were incubated with Akt inhibitor IV (1.25 µM; EMD Millipore, Billerica, MA), PI3K inhibitor (LY294002; 20 µM; Sigma-Aldrich; St. Louis, MO), EGFR inhibitor (AG1478; 1 µM; EMD Millipore), a broad spectrum ADAM inhibitor (GM6001; 10 µM; EMD Millipore) (all 1 h prior to challenge with T. gondii ), Pertussis Toxin (PTx; 100 ng/ml; EMD Millipore; 4 h prior to challenge), leupeptin (10 µM; EMD Millipore) and pepstatin (10 µM; EMD Millipore; both 1 h after challenge with T. gondii ), 3-methyl adenine (3MA; 10 mM; Sigma Chemical) and rapamycin (1 µM; EMD Milipore; both 2 h after challenge with T. gondii ) or vehicle.

Techniques: Incubation, Light Microscopy, Transfection, Fluorescence, Microscopy, Electron Microscopy, Expressing, Infection